We are investigating the effect of the 5' untranslated region of mRNAs on the stability of spliced and unspliced messages. We have ligated the 5' end of the HSV-TK gene extending from 15 nucleotides before the initiator AUG codon to about 550 nucleotides befor the AUG into the 5' junction between the SV40 sequences and the bacterial chloramphenicol acetyltransferase (CAT) sequences in the recombinant plasmid pSV2-CAT. We have also removed a fragment from pSV2-CAT containing the SV40 t intron and splice sites, but not disturbing CAT sequences. These three recombinant plasmids have been used to transfect CV-1 cells and extracts from the cells analyzed for CAT activity. Cells transfected with the parent pSV2-CAT show considerable CAT activity as do those transfected with DNA lacking the t intron, while those transfected with the recombinant containing the S' end of the HTR gene show reduced activity.